Separation of erythrocyte stroma from lysing medium and hemoglobin with acrinol

ABSTRACT

A process for enhancing the separation of erythrocyte stroma from a suspension of erythrocyte lysing medium and hemoglobin which comprises A. LYSING ERYTHROCYTES AND B. CONTACTING THE LYSED ERYTHROCYTES WITH ACRINOL.

Unite tates Dahlgren et a1.

Sept. 2, 1975 SEPARATION OF ERYTHROCYTE STROMA FROM LYSING MEDIUM AND HEMOGLOBIN WITH ACRINOL Inventors: Donald A. Dahlgren, Portage; John W. Nelson, Kalamazoo, both of Mich.

Assignee: The Upjohn Company, Kalamazoo,

Mich.

Filed: June 4, 1973 Appl. No.: 367,022

Related US. Application Data Division of Ser. No. 165,697, Oct. 23, 1971, Pat. No. 3,745,155.

US. Cl. 424/12; 210/DIG. 23; 260/112 B; 424/11; 424/13; 424/85; 424/88; 424/101 Int. Cl. A61K 35/18; BOlD 21/00; GOIN 31/O0;GO1N 33/16 Field of Search 260/112 R, 1 12 B; 424/8, 424/1l,12,13, 85, 88,101;210/DIG.23,

Primary Examiner-Albert T. Meyers Assistant ExaminerA. P. Fagelson Attorney, Agent, or FirmMartin B. Barancik; Roman Saliwanchik 5 7] ABSTRACT A process for enhancing the separation of erythrocyte stroma from a suspension of erythrocyte lysing medium and hemoglobin which comprises a. lysing erythrocytes and b. contacting the lysed erythrocytes with acrinol.

1 Claim, N0 Drawings SEPARATION OF ERYTI'IROCYTE STROMA FROM LYSING MEDIUM AND I-IEMOGLOBIN WITH ACRINOL This application is a divisional of co-pending patent application Ser. No. 165,697 filed July 23, 1971, now U.S. Pat. No. 3745155.

This invention relates to the purification of gamma globulin. More specifically, this invention relates to a process of adsorbing certain antibodies to human blood elements which contaminate horse anti-human lymphocytic gamma globulin.

BACKGROUND OF THE INVENTION Gamma globulin is well known as the blood protein fraction which has the bulk of antigen-inactivating agents known as antibodies. These antibodies aid a host in combatting disease, conditions and the like. Antibody formation is stimulated by the presence of antigens in the animal. In the production of anti-human lymphocytic gamma globulin, i.e., ALG, in an animal, preferably a horse, other human substances not fully separated from the lymphocytic tissue are injected into the animal as well. Of particular importance are other blood and tissue elements, for example, erythrocytes, thrombocytes, albumin, alpha and beta globulin, etc. Antibodies against these foreign elements, as well as antibodies against the lymphocytes, are produced. If gamma globulin obtained from a horse thus treated were administered to a human, troublesome antigenantibody interaction due to horse anti-human erythrocyte and thrombocyte antibodies, for example, as well as the desired antihuman lymphocyte effect, can occur. In order to have the horse anti-human lymphocyte gamma globulin as specific as possible, the undesirable antibodies within the ALG are commonly adsorbed and usually removed from the ALG by contacting the gamma globulin with human erythrocytes or their stroma, human thrombocytes or their stroma, and/or absorbed with human serum or plasma. Not only are undesirable antibodies which are immunologically different from the anti-human lymphocyte gamma globulin thus inactivated, but antibodies cross reactive to various human blood antigens are removed in this manner as well.

Although these art procedures are theoretically simple, a number of significant problems are encountered in practice. When employing serum or plasma to absorb contaminating antibodies, the antigen-antibody complexes usually do not precipitate. Additionally, there is no practical means of separating the gamma globulin contained in the absorbing plasma or serum from the ALG. The gamma globulin of the absorbing plasma or serum contaminates the final product. Consequently, not only is the ALG administered to the pa tient but also a large quantity of unnecessary and possibly antigenic protein. As well as the difficulty of the body metabolizing the large quantity of extra protein, the administration of such a mixture of proteins appears to have caused immune complex disease in some cases. Additionally, a further disadvantage of using human whole blood serum or plasma for absorbing un desirable antibodies is the time consuming assays required to assess the level of contaminating protein remaining in the ALG.

In another prior art ALG purification step, erythrocyte stroma are employed to adsorb the anti-human erythrocyte antibodies from the gamma globulin. Prior to contacting the ALG with the erythrocyte stroma, the contaminating stroma lysing medium and hemoglobin, that is, the menstruum, must first be removed from the stroma. This is generally done by separating the stroma from the contaminating liquid by centrifugation and washing. However, much difficulty is encountered because the stroma, particularly stroma from outdated blood, are not readily separated from the menstruum.

These problems and other problems mentioned hereafter are solved by the procedure of this invention.

BRIEF SUMMARY OF THE INVENTION In accordance with this invention, we have discovered a process for purifying a gamma globulin raised against a heterologous antigenic material, said antigenic material contaminated with antigenic blood components from the same species animal source as the antigenic material, comprising contacting a solution of said gamma globulin with solid plasma protein essentially free of gamma globulin and obtained from the same species animal source as the said antigenic material. Contaminating antiblood component antibodies of the said solution of gamma globulin are adsorbed upon the said solid plasma protein. The solution of a purified gamma globulin is then separated from the solid material. 1:1:

Another aspect of this invention is contacting a solution of gamma globulin witherythrocyte stroma in addition to solid plasma protein. A still further aspect of this invention is the addition of acrinol to a suspension of lysed erythrocytes to enhance the separation of the stroma from the menstruum.

Although the invention is disclosed in detail with regard to horse anti-human lymphocyte gamma globulin, the invention is not so restricted. Other suitable antigenic materials include carcino-embryonic tissue, for example. When the antigenic material is lymphocytic, suitable sources include spleen lymphocytes, thymocytes, blood lymphocytes, thoracic duct lymphocytes, and cultured human lymphocytes, preferably thymocytes. The animal from which the antigenic material is taken include chimpanzee, monkey, rabbit, rat, dog, pig, etc., preferably the human. The animal whose gamma globulin is raised against the antigenic material includes horse, goat, cow, sheep, rabbit, chimpanzee, monkey, rat, preferably the horse.

When the antigenic materials are lymphocytes, the gamma globulin has immunosuppressive effects when administered to the species of animal from whom the antigenic material was taken. The gamma globulin is useful, for example, in enhancing survival of homologous and heterologous organ and skin transplants, which include kidney, liver, heart, lung, bone marrow, and skin in humans and like transplants in animals such as chimpanzee and monkey.

DETAILED DESCRIPTION Gamma globulin can be essentially separated from blood serum or plasma, interchangeable for the purposes of this specification and claims, by variety of conventional means. Examples of reagents which effect this separation include neutral salts including sulfates, thiosulfates, phosphates, halogen salts of alkali metals, ammonium or magnesium; metal ions employed in suitable concentrations, non-ionizable polymers of high molecular weight, polyanions, water soluble organic solvents, e.g., ethanol, methanol, isopropanol, organic cations, e.g., acrinol and other acridines including tryplaflavine, S-aminoacridine (Clinica Chimica Acta 6 (1961) 782-793). The critical factor common to all these methods is that the gamma globulin has been essentially separated from the bulk of the serum protein and thus it cannot contaminate the heterologously raised gamma globulin when the solid plasma protein is employed to adsorb undesired antibodies. The process will be exemplified with regard to acrinol which has the further advantage of effectively separating gamma globulin from whole blood as well as serum or plasma. This feature is particularly useful in separating gamma globulin from cow whole blood since cow erythrocytes do not separate as rapidly as human erythrocytes.

Blood plasma from the animal species used as the source of the antigen is treated with an aqueous solution of acrinol containing from about 0.1 to about percent acrinol, to precipitate blood proteins other than gamma globulin. Among these proteins are albumin, alpha and beta globulin, etc. The plasma is at its normal pH of about 7 to 8, although it can be adjusted to about pH 7.6 by the addition of a suitable reagent such as 1N sodium hydroxide, 0.8M sodium bicarbonate, or lM sodium carbonate or 1N l-lCl or acetic acid. The precipitation is usually done at about 25 C., although higher or lower temperatures are operable. From 3.5 to 5 volumes, preferably about 4 volumes, of 0.4 percent aqueous acrinol solution is used for each volume of plasma. Precipitation is allowed to go to completion with settling and flocculation during standing, preferably at a temperature of about 4 C. from about minutes up to as long as overnight or longer. The precipitate is separated from the supernatant containing the gamma globulin, illustratively by filtration, decantation or centrifugation. The precipitate contain ing the acrinol-insoluble blood plasma protein is washed with a 0.1% to 5 percent aqueous solution of acrinol. The washed precipitate is then transferred to a solution of gamma globulin raised against the antigen from the heterologous source and stirred for a few minutes to several hours at about 4 C. to about 40 C., preferably about C., so that the anti-plasma protein antibodies contained in the solution of gamma globulin are adsorbed upon the solid plasma protein. A sufficient quantity of gamma globulin separating reagent is maintained in the suspension so that the multiphase adsorption condition exists. The solid plasma protein with adsorbed anti-plasma protein antibodies is then separated by, for example, decantation, filtration, or centrifugation. The adsorption process can be repeated as many times as necessary to reduce the contaminating antibodies to the desired level. The quantity of solid plasma protein obtained from 0.0l to 5 or more liters of plasma is used to adsorb the anti-plasma protein antibodies from heterologously raised anti-human lymphocyte gamma globulin obtained from 1 liter of plasma. This method of adsorption is additionally advantageous when acrinol is employed as the gamma globulin separating reagent, because hepatitis virus appears to be precipitated along with the plasma protein, thereby minimizing the possibility of hepatitis virus contamination of the final product.

The gamma globulin raised against the antigenic material can be conveniently prepared by.methods well known in the art. A species of animal, preferably the horse, is inoculated illustratively with lymphoid tissue from a different species of animal, preferably the human. The lymphocytes are prepared by methods known in the art, for example, by rate zonal centrifugation, to obtain separation of lymphocytes and platelets from granulocytes and erythrocytes. Thereafter, isopycnic zonal sedimentation can be used to separate lymphocytes from the platelets. Although a reasonably pure population of lymphocytes is obtained in this manner, the resulting lymphocyte preparation is still contaminated by various blood elements, most importantly by human erythrocytes and other antigenic blood components. The animal is then inoculated with the lymphocyte preparation. After a suitable length of time, the animal is bled and the gamma globulin fraction isolated by methods previously disclosed in this specification.

As stated previously, when preparing a purified gamma globulin intended for internal use it is also desirable to remove anti-erythrocyte antibodies from the gamma globulin. This is conveniently done by contacting the solution of gamma globulin with erythrocytes or preferably with erythrocyte stroma. Preparation of the stroma for contact with the gamma globulin is by methods well known in the art except for a novel improvement in one of the isolation steps which is as follows. After lysing the erythrocytes and before contacting the gamma globulin with the stroma, the menstruum must be separated from the stroma as well as possible. The addition of small quantities of acrinol to the lysed erythrocytes enhance the separation of the stroma from the menstruum and only two or three washings and centrifugations at relatively low velocities of 2000 r.p.m. for 10 to 15 minutes suffice for the separation as compared to the 10 or more washings and centrifugations at 3,000 r.p.m. or more for 30 minutes practiced by the art. Indeed satisfactory separation is often achieved by the subject improvement by mere settling overnight in the cold, for example, followed by decantation. The stroma are sufficiently free of hemoglobin when the characteristic color of the suspending fluid is essentially gone as measured by sight or examined spectrophotometrically at 555 nm. The amount of acrinol to be employed varies from about 1 to 5 g/l of original packed erythrocytes, preferably about 3 to 5 g/l.

The preliminary steps of preparing stroma are those known in the art. To the erythrocyte precipitate is added an equal volume of 0.9 percent NaCl solution. The suspension is then mixed and centrifuged, followed by the removal of the buffy coat. The washed erythrocytes are then lysed by any standard lysing procedure, for example, digitonin, water, freezing and thawing. The stroma are separated from the suspension with the aid of acrinol, as previously disclosed. it should be further pointed out that choice of an acrinol solution of appropriate tonicity allows the erythrocyte stroma to be lysed and separated by the single reagent, thereby eliminating a separate lysing step.

The stroma are then added to the solution of gamma globulin raised against the heterologous antigen so that adsorption of the anti-erythrocyte antibodies with the stroma can be effected. This adsorption is carried out as many times as necessary to adsorb the antierythrocyte antibodies. The quantity of stroma obtained from about 0.01 to 5 or more liters of packed erythrocytes are used to adsorb the antierythrocyte antibodies from heterologously raised gamma globulin obtained from 1 liter of plasma. The multiphase adsorption is maintained by an appropriate quantity of the gamma globulin separating agent.

The stroma adsorption can be carried out prior to, concurrently with, or after the plasma protein adsorption.

Following are specific examples setting forth methods of preparing and using the invention described in this application. These examples are not meant to limit, but are employed merely to exemplify the invention.

EXAMPLE 1 Preparation of Anti-Human Lymphocyte Gamma Globulin Human lymphocytes are prepared by any convenient manner known in the art and a horse is injected with these lymphocytes. When the desired antibody titer is attained, the horse is bled, the serum separated from the whole blood and the serum is adjusted to a pH of about 7.6 by the addition of 0.8M sodium bicarbonate. An aqueous solution containing about 0.4 percent acrinol is added slowly to the serum, with stirring, to precipitate blood proteins other than the gamma globulin. An excess of the acrinol solution is employed to insure complete precipitation. Precipitate settling is allowed to continue overnight at a temperature of about 4 C. The supernatant containing the gamma globulin is decanted from the precipitate,

EXAMPLE 2 Absorption of Human Anti-Plasma Protein Antibodies with Whole Serum by Prior Art Process To a horse anti-human lymphocyte gamma globulin solution prepared as in Example l is added an equal volume of human whole serum. This whole serum inactivates the human blood protein antibodies in the horse antilymphocyte gamma globulin. However, the human gamma globulin is mixed with the horse anti-human lymphocyte gamma globulin and the two can only be separated with very great difficulty if at all.

EXAMPLE 3 Adsorption of Human Anti-Plasma Protein Antibodies with Solid Plasma Protein A sufficient quantity of an aqueous solution containing about 0.4 percent acrinol is added slowly to human plasma, previously adjusted to a pH of about 7.6 with 0.8M sodium bicarbonate, with stirring in order to precipitate the acrinol-insoluble proteins, The resulting precipitate is separated from the supernatant by centrifugation. Solid plasma protein obtained from 0.2 liters of human plasma is added to a solution of ALG obtained from l liter of plasma prepared as in Example 1. This resulting suspension is stirred continually for a period of 3 hours at room temperature, then refrigerated overnight. The supernatant containing the ALG purified from contaminating human anti-plasma protein antibodies is then separated by centrifugation at 2000 r.p.m. for 10 minutes. This ALG is essentially uncontaminated with harmful human blood plasma proteins or soluble antigen antibody complexes.

EXAMPLE 4 Adsorption of Human Anti-Erythrocyte Antibodies by Prior Art Process To a volume of packed human erythrocytes is added an equal volume of aqueous 0.9 percent M NaCl. The solution is mixed and centrifuged at 3000 r.p.m. for 30 minutes at 2 C. and the supernatant and buffy coat discarded. To each liter of packed erythrocytes is added 10 volumes of distilled water and the cells are lysed by standing for 30 minutes. The stroma are washed and centrifuged l0 times at 3000 r.p.m. for 30 minutes to reduce the hemoglobin to satisfactory levels. Some stroma are lost in the washing process. Stroma from 0.1 volume of packed erythrocytes are added to the ALG obtained from 1 liter of plasma as prepared in Example 1 and stirred for 3 hours at 2 C. The stroma are separated from the ALG solution by centrifugation and the supernatant treated with additional stroma two more times to reduce antierythrocyte titer to an acceptable level.

EXAMPLE 5 Adsorption of Human Anti-Erythrocyte Antibodies from ALG with Stroma Prepared by the Addition of Acrinol The process of Example 4 is repeated to a point wherein the erythrocytes are lysed with distilled water. At this point the lysed erythrocyte solution is diluted with 2 /2 volumes of water calculated on the basis of the original packed erythrocyte volume. Five grams of acrinol for each liter of original packed erythrocyte volume is then added. This suspension is stirred for one hour at room temperature and placed in the refrigerator overnight. The suspension is centrifuged at 2000 r.p.m. for 10 minutes and the ALG solution separated from the stroma. Two additional fresh stroma adsorptions are carried out in the above manner to reduce the anti-erythrocyte titer to a satisfactory level.

We claim:

1. A process for enhancing the separation of erythrocyte stroma from a suspension of erythrocyte lysing medium and hemoglobin which comprises a. lysing erythrocytes and b. contacting the lysed erythrocytes with acrinol,

* l l l 

1. A PROCESS FOR ENCHANCING THE SEPERATING OF ERYTHROCYTE STROMA FROM A SUSPENSION OF ERYTHROCYTE LYSING MEDIUM AND HEMOGLOBIN WHICH COMPRISES A. LYSING ERYTHROCYTES AND B. CONTACTING THE LYSED ERYTHROCYTES WITH ACRINOL. 